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Journal of Histochemistry and Cytochemistry, Vol. 50, 863-874, June 2002, Copyright © 2002, The Histochemical Society, Inc.

ARTICLE

Optimization of Immunogold Labeling TEM: An ELISA-based Method for Evaluation of Blocking Agents for Quantitative Detection of Antigen

Ramandeep Kaura, Kanak L. Dikshita, and Manoj Rajea
a Institute of Microbial Technology, Chandigarh, India

Correspondence to: Manoj Raje, Inst. of Microbial Technology, Sector 39A, Chandigarh 160036, India. E-mail: manoj@imtech.res.in

We developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy. Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA and the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Ability to minimize NSB was also evaluated by dot-blot and Western blotting methods. The results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM. Existing methods could not provide an accurate picture of the ability of various reagents to suppress background labeling. The sensitivity of detection of antigens by immunoelectron microscopy depends on the assay procedure being optimized to obtain the highest possible signal along with as low a background (noise) as possible. Our study indicated that an ELISA-based evaluation of various blocking agents could help in the rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigens by TEM. (J Histochem Cytochem 50:863–873, 2002)

Key Words: antigen detection, antigen quantitation, transmission electron, microscopy, nonspecific binding, artificial immunospecimen, ELISA, blocking, signal-to-noise ratio, immunogold labeling


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